Confocal microscope with double-objective system

ABSTRACT

A double-objective system for a microscope, wherein an objective is provided for focusing object illumination light onto an object point and an objective is provided for collecting light that proceeds from the object point. The optical axes of the two objectives are inclined relative to one another, so that the observation direction and the illumination direction are at an angle relative to one another. The two objectives are combined at a shared holder to form a double-objective constructional unit for guiding light from an object illumination source through a light passage opening, wherein, further, a light deflection system is provided, which shapes the illumination beam path and/or the observation beam path in such a way that the observation light coming from the observation objective exits the double-objective constructional unit through the light passage opening. The double-objective constructional unit can therefore be used in a conventional microscope construction.

BACKGROUND OF THE INVENTION

a) Field of the Invention

The invention is directed to a confocal microscope with adouble-objective system comprising a first objective, which is providedfor focussing light from an object illumination source onto an objectpoint in an illumination beam path, and a second objective which isprovided for collecting light that proceeds from the object point in anobservation beam path, wherein the observation direction of the secondobjective is at an angle to the illumination direction of the firstobjective.

b) Description of the Related Art

A scanning microscope with a dual-objective system of the type mentionedabove is known from DE 43 26 473 A1. In this known scanning microscope,collimated laser light is focussed through the first objective in theillumination beam path onto an object point. The second objective isarranged relative to the first objective in such a way that the opticalaxes of the two objectives are perpendicular to one another and theobject point illuminated by the first objective lies in the principalfocus of the second objective, so that the observation direction of thesecond objective is at a right angle to the illumination direction ofthe second objective. The observation light proceeding from the objectpoint and collected by the second objective is focussed onto a pinholediaphragm by a lens arranged following the second objective in theobservation beam path, wherein a light detector measures the intensityof the light passing through the pinhole diaphragm. A correspondingimage can then be generated from the measurement information of thelight detector by scanning an object area.

Further, an incident illumination for stereo microscopes known from DE94 08 066 U1 illuminates the object to be observed from the direction ofthe observation optics as far as possible. It is realized in such a waythat the illumination arrangement forms a constructional unit with themicroscope body and accordingly with the microscope objective acting asdetector arrangement.

This illumination arrangement differs from the double-objective systemaccording to the invention in particular in that it is not suitable foruse in a confocal microscope and in that it illuminates as far aspossible from the detection direction, whereas in the double-objectivesystem according to the invention a right angle is realized as far aspossible between the illumination direction and the detection direction.

The scanning microscope known from DE 43 26 473 A1 already has anextremely high resolution. This is achieved in particular in that theconfocal volume detected in the in-focus or focal range is minimized dueto the intersection of the optical axes of the illumination beam pathand observation beam path at a right angle in the object point. When theillumination volume in the object area is considered in a roughapproximation as an ellipsoid stretched along the illumination axis andthe observation volume of the second objective in the object area isconsidered as an ellipsoid stretched along the optical axis of thesecond objective, a superposition of these ellipsoids in the focal rangeor in the object point region gives a correspondingly smaller confocalvolume which substantially corresponds to the intersection volume of theillumination volume and observation volume. The smaller the confocalvolume considered in this way, the better the resolution of themicroscope.

An analogous manner of considering assumes that the intensitydistribution of the object illumination light in the focal range of thefirst objective is described by the illumination point spread function,as it is called, and that the detection probability for the lightproceeding from the focal range by means of the second objective whenusing a detection pinhole is described by the observation (detection)point spread function.

The resulting confocal point spread function KPVF of a confocalmicroscope is the product of the illumination point spread function andthe observation point spread function. The more extensive the KPVF, thepoorer the resolution of the microscope. As a result of the nowvirtually perpendicular arrangement of the illumination point spreadfunction and the observation point spread function relative to oneanother, the comparatively large expanse of the illumination pointspread function along the optical axis of the first objective iscompensated by the small expanse of the illumination point spreadfunction along this axis, so that an approximately equally goodresolution is achieved along all three spatial axes. Therefore, a verygood resolution capacity is achieved because of the illumination andobservation beam paths which intersect at a right angle in the objectpoint.

According to DE 43 26 473 A1, a special construction with completeseparation of the observation beam path from the illumination beam pathis provided for realizing the beam paths for illumination andobservation which intersect in the object point. A special constructionof this type requires a comparatively large amount of room in the twodirections of the beam paths for illumination and observation at rightangles to one another and is accordingly more difficult to manage thanconventional scanning microscopes in which the illumination beam pathand the observation beam path coincide and the focussing of the objectillumination light and the collection of the observation light comingfrom the object point are effected through the same objective.

OBJECT AND SUMMARY OF THE INVENTION

It is an object of the present invention to achieve the foregoing statedobjects by an optical light microscope without deviating from previousconstruction designs.

In accordance with the present invention, a double-objective system fora microscope includes a first objective which is provided for focussinglight of an object illumination source onto an object point in anillumination beam path, and a second objective which is provided forcollecting light that proceeds from the object point in an observationbeam path. The observation direction of the second objective is at anangle (α) to the illumination direction of the first objective. The twoobjectives are attached to a shared holder so as to form adouble-objective constructional unit containing both objectives, thedouble-objective constructional unit contains a light passage openingfor guiding light from the object illumination source and furthercontains a light deflection system for shaping at least one of theillumination beam path and the observation beam path so that observationlight coming from the second objective exits the double-objectiveconstructional unit through the light passage opening.

An embodiment example of the invention is explained more fullyhereinafter with reference to the drawing.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 shows a schematic sectional view of an embodiment example of adouble-objective system according to the invention.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention, in accordance with its object, indicates a methodsuch as the optical principle known from DE 43 26 473 A1 which isapplicable in an optical light microscope without deviating from theconstruction design of a conventional microscope.

For this purpose, it is suggested, according to the invention, that thetwo objectives are attached to a common holder so as to form a cohesivecomponent group or constructional unit containing both objectives,wherein the double-objective constructional unit contains a lightpassage opening for guiding light from an object illumination source andfurther contains a light deflection system which shapes the illuminationbeam path and/or the observation beam path in such a way that theobservation light coming from the second objective exits thedouble-objective constructional unit through the light passage opening.

Since the light enters and exits through a common light passage opening,the double-objective system, according to the invention, can be used asan objective system which is compatible with the beam path for amicroscope, in particular a scanning microscope, which is configured ina conventional manner in other respects and is designed for illuminationand observation through the same objective, wherein, in the conventionalmicroscope, it can be substituted for the conventional individualobjective.

Therefore, the optical principle known from DE 43 26 473 A1 forachieving an extremely high resolution in all three spatial directionscan be realized in combination with a conventional microscopeconstruction.

The two objectives and the light deflection system are preferablyaccommodated in a common housing which has an opening for theintroduction of light of an object illumination source and for guidingout the observation light.

The housing is preferably attachable to the objective holder of anotherwise conventional microscope, especially so as to be rotatableabout the axis of the holder, in order to integrate the illuminationbeam path of the double-objective constructional unit in theillumination beam path of the microscope and to integrate theobservation beam path of the double-objective constructional unit in theobservation beam path of the microscope.

With respect to the aimed for high resolution, the angle between theillumination direction of the first objective and the observationdirection of the second objective is approximately 90°. However, theadvantages of the invention are also still achieved to a sufficientextent if the angle deviates not too substantially from 90°.

The terms illumination direction and observation direction refer,respectively, to the direction of the outlet beam path of the firstobjective and to the direction of the inlet beam path of the secondobjective.

In accordance with a further development of the invention, it can beprovided that the objectives are adjustable relative to one another inorder to be able to selectively change the angle between theillumination direction and observation direction. For example, if theangle is set at somewhat less than 90°, the specimen space in front ofthe double-objective constructional unit is less narrowly restrictedwithout making great compromises with respect to the desired resolution.

The objectives preferably have the same numerical aperture, especially alarge numerical aperture, and can have conventional lens elements. Theobjectives should preferably have a long working distance.

In order to reduce the angle between the optical axes of the twoobjectives, lens elements of the objectives can be ground. The field ofview of the individual objectives is accordingly no longer round.However, when the double-objective system is used in a scanningmicroscope with beam scanning, this has no influence on the imagingcharacteristics, since, in each case, the beam is scanned in only onedimension which then extends perpendicular to the optical axes of theobjectives.

Further, the fact that the double-objective constructional unitaccording to the invention can be used in a conventional scanningmicroscope construction results in the substantial advantage that thebeam movement means and object movement means required for scanning(beam scanning and/or object scanning) can also be of conventionaldesign and need not be specially adapted. The beam scanning is usuallycarried out vertical to the optical axes of the two objectives. In thisway, it is ensured in the case of beam scanning that the illuminationpoint and observation point are moved jointly.

The double-objective constructional unit according to the inventionadvantageously allows the use of an immersion medium when used in ascanning microscope.

The light deflection system of the double-objective constructional unitcan contain mirrors, dichroic mirrors, beam splitters or optical fibers(60 in FIG. 1), for example.

At least some optical elements of the light deflection system arepreferably fastened in the housing so as to be movable for adjustmentpurposes and/or exchangeable, as needed. This is also true for theobjectives.

The double-objective constructional unit according to the invention canbe handled in a simple manner essentially like a conventional objectivein a conventional microscope and can be used in a fluorescencemicroscope in which the fluorescence dyes are excited by single-photonor multi-photon absorption. For observation of fluorescent light,suitable optical elements affecting the wavelength of the light can beprovided. Optical elements of this kind, for example, spectral filters,can be used in the beam paths for illumination and observation whichextend separately within the double-objective constructional unit. Onthe other hand, elements of this kind can also be introduced at alocation at which the beam paths run together.

Alternatively, the double-objective constructional unit according to theinvention can also be used in a microscope which operates by scatteredlight. For observation of scattered light, suitable optical elementsaffecting the polarization direction of the light can be provided.

Polarizers of the type mentioned above can be used, for example, in thebeam paths for illumination and observation which extend separately inthe double-objective constructional unit. However, as an alternative, itis also possible to use them at a location at which the two beam pathsrun together.

The double-objective system according to the invention can beconstructed in such a way that illumination and observation can beexchanged with respect to the two beam paths mentioned above. A furtherpossibility consists in that only one or the other beam path, as needed,can be used in a conventional manner simultaneously as illumination beampath and observation beam path, for example, in order to carry outvisual observation of the object point by means of an eyepiece system.

Referring now to the drawing, the double-objective system of FIG. 1 isshown as a cohesive structural component group 1 (double-objectiveconstructional unit) which can be handled as a unit. Thedouble-objective constructional unit 1 has a housing 3 with a lightpassage opening 5 which is enclosed by a mounting or frame 7. The frame7 of the double-objective constructional unit 1 is to be connected tothe objective holder 9, indicated schematically, of a microscope 11. Thelight path, indicated in dashed lines, passes within objective holder 9and frame 7, and the light passage opening 5 is centered with respect tothe optical tube axis 13, wherein the double-objective constructionalunit 1 is optionally rotatable about the optical axis 13.

The double-objective constructional unit 1 has an illumination beam path15 which forms part of the overall illumination beam path of themicroscope 11. As in a reflected light microscope, the light coming froman object illumination source 53 of the microscope 11 via a beamsplitter 47 and mirror 45 enters the objective system 1 along theoptical axis 13 through the opening 5. The object illumination light isthen reflected laterally at the surface 17 of the beam splitter 19 of alight deflection system arranged in the housing 3 and strikes thedeflecting mirrors 21 and 23 in succession, so that the light reflectedby the deflecting mirror 23 finally enters the illumination beam path 15through a first objective 25. The first objective 25 serves to focus thelight from the object illumination source onto an object point 27 of anobject resting on an object carrier 29.

As can be seen from FIG. 1, the optical axis 31 of the first objective25 is inclined at an angle β₁ relative to the optical tube axis 13 ofthe microscope 11.

In order to observe the object point 27, the double-objectiveconstructional unit 1 has a second objective 33 in the housing 3, whichsecond objective 33 is arranged in the observation beam path 35 andcollects light coming from the object point 27. A deflecting mirror 37is provided at the light outlet side of the second objective 33, whichdeflecting mirror 37 reflects the observation light collected by thesecond objective 33 laterally to an additional deflecting mirror 39. Theobservation light reflected by the deflecting mirror 39 then passesthrough the beam splitter 19 with a small offset and finally passesalong the optical axis 13 through the opening 5 via mirror 45 and beamsplitter 47 to an observation system (not shown) or to the detector 51,shown in the drawing, with a pinhole diaphragm 49 of a light detectionsystem of the microscope 11, which pinhole diaphragm 49 is arranged infront of the detector 51.

The optical axis 41 of the second objective 33 is inclined at an angleβ₂ relative to the optical axis 13, wherein the angle β₂ is preferablyequal to the opposite angle β₁.

The optical axes 31 and 41 of the two objectives 25 and 33 enclose anangle α which is somewhat less than 90° in the example shown in FIG. 1,but which can also be exactly 90° in an alternative embodiment form.

By arranging the observation objective 33 relative to the illuminationobjective 25 in the manner mentioned above, the double-objectiveconstructional unit 1 according to the invention achieves the advantagesof extremely high resolution as in the scanning microscope according toDE-OS 43 26 473, and it is possible to make recordings with the highestresolution that a far-field light microscope can have without the use ofinterference. Further, the double-objective constructional unit 1according to the invention can be used in a conventional scanningmicroscope which has a common beam path for illumination and observationat the point of intersection with the objective by means of aconventional objective, since the illumination beam path and theobservation beam path of the double-objective constructional unit 1converge essentially along the axis 13 in the area of the light passageopening 5 and accordingly in the area of connection to the conventionalmicroscope, so that the double-objective constructional unit 1 accordingto the invention has a connection compatibility with respect to theillumination beam path and observation beam path of the conventionalmicroscope construction.

Since the two objectives 25 and 33 and the light deflection system 19,21, 23, 37, 39 are accommodated in a common housing 3, thedouble-objective constructional unit 1 according to the invention can behandled essentially as a conventional individual objective in aconventional scanning microscope. The double-objective constructionalunit 1 can preferably be attached to the microscope construction 11 soas to be removable, so that it can be exchanged for another objective,if required, for example, a conventional individual objective.

The objectives 25 and 33 preferably comprise conventional objectivelenses. In particular, the objectives 25 and 33 have the sameconstruction and the same numerical aperture, wherein the numericalaperture should be as large as possible, and wherein the objectives 25and 33 should preferably have a long working distance and a small fieldof view.

In the embodiment example according to FIG. 1, the optical elements inthe illumination beam path 35 (especially the deflecting mirrors 37 and39) are arranged so as to be stationary with respect to the housing 3,so that the observation point can be fixed and can serve as a referencefor the adjustment of the illumination point. In this case, theadjustment of the illumination beam path requires five degrees offreedom which can be made available, for example, by the deflectingmirrors 21 and 23 with two degrees of freedom, respectively, and by theobjective 25 with one degree of freedom.

An object carrier stage which is movable in a controlled manner by meansof a scanning control and which enables object scanning throughcontrolled movement of the object relative to the microscope isdesignated in FIG. 1 by 43. The object carrier stage 43 can preferablycarry out translational movements and rotational movements in that it iscontrolled by the scanning control.

The double-objective constructional unit 1 according to the inventioncan also be used in a microscope with beam scanning in which a scanningunit (not shown) of the conventional microscope 11 moves the commonillumination and observation beam path in such a way that theillumination point and the observation point are moved relative to theobject under controlled conditions, which results in a substantialadvantage with respect to speed. The object scanning and beam scanningoperation modes can also be combined if required.

While the foregoing description and drawings represent the presentinvention, it will be obvious to those skilled in the art that variouschanges may be made therein without departing from the true spirit andscope of the present invention.

What is claimed is:
 1. A double-objective system for a microscope,comprising:a first objective which is provided for focussing light of anobject illumination source onto an object point in an illumination beampath; a second objective which is provided for collecting light thatproceeds from the object point in an observation beam path, anobservation direction of the second objective being at an angle (α) toan illumination direction of the first objective; said two objectivesbeing attached to a shared holder so as to form a double-objectiveconstructional unit containing both objectives; said double-objectiveconstructional unit containing a light passage opening for guiding lightfrom said object illumination source and further containing a lightdeflection system for shaping at least one of the illumination beam pathand the observation beam path so that observation light coming from thesecond objective exits the double-objective constructional unit throughthe light passage opening.
 2. The double-objective system according toclaim 1, wherein the shared holder has a housing which contains the twoobjectives and the light deflection system and has a housing openingforming the light passage opening.
 3. The double-objective systemaccording to claim 2, wherein the housing is rotatable attached to anobjective holder of a microscope, aid housing being rotatable about anaxis of the objective holder, in order to integrate the illuminationbeam path of the double-objective constructional unit in an illuminationbeam path of the microscope and in order to integrate the observationbeam path of the double-objective constructional unit in an observationbeam path of the microscope.
 4. The double-objective system according toclaim 1, wherein the angle (α) between the illumination direction of thefirst objective and the observation direction of the second objective isapproximately 90°.
 5. The double-objective system according to claim 1,wherein at least one of the first objective and the second objective isheld so as to be movable for selectively changing the angle (α) betweenthe illumination direction and the observation direction.
 6. Thedouble-objective system according to claim 1, wherein an optical axis ofthe first objective extends at a first angle of inclination (β₁)relative to an axis of the light passage opening, and wherein lightdeflecting elements of the light deflection system are provided on alight inlet side of the first objective, which light deflecting elementsdeflect light of the object illumination source incident along the axisof the light passage opening for passage through the first objective. 7.The double-objective system according to claim 1, wherein an opticalaxis of the second objective extends at an angle of inclination (β₂)relative to an axis of the light passage opening, and wherein lightdeflecting elements of the light deflection system are provided on alight outlet side of the second objective, which light deflectingelements deflect observation light coming from the second objective, sothat the observation light exits the double-objective constructionalunit along the axis of the light passage opening.
 8. Thedouble-objective system according to claim 1, wherein the lightdeflection system contains optical light-conducting fibers.
 9. Thedouble-objective system according to claim 1, wherein the two objectiveshave substantially the same numerical aperture.
 10. Microscope with adouble-objective system, comprising:a first objective which is providedfor focussing light of an object illumination source onto an objectpoint in an illumination beam path; a second objective which is providedfor collecting light that proceeds from the object point in anobservation beam path, an observation direction of the second objectivebeing at an angle (α) to an illumination direction of the firstobjective; said two objectives being attached to a shared holder so asto form a double-objective constructional unit containing bothobjectives; said double-objective constructional unit containing a lightpassage opening for guiding light from said object illumination sourceand further containing a light deflection system for shaping at leastone of the illumination beam path and the observation beam path so thatobservation light coming from the second objective exits thedouble-objective constructional unit through the light passage opening.11. The microscope according to claim 10, wherein said microscope is afluorescence microscope.
 12. An arrangement for the observation ofscattered and/or reflected light, comprising:a microscope having:a firstobjective which is provided for focussing light of an objectillumination source onto an object point in an illumination beam path; asecond objective which is provided for collecting light that proceedsfrom the object point in an observation beam path, an observationdirection of the second objective being at an angle (α) to anillumination direction of the first objective; said two objectives beingattached to a shared holder so as to form a double-objectiveconstructional unit containing both objectives; said double-objectiveconstructional unit containing a light passage opening for guiding lightfrom said object illumination source and further containing a lightdeflection system for shaping at least one of the illumination beam pathand the observation beam path so that observation light coming from thesecond objective exits the double-objective constructional unit throughthe light passage opening, said arrangement being adapted to observescattered and/or reflected light.